DESCRIPTION: The PI proposes that a novel paracrine system based on purinergic signaling allows for intraepithelial communication between the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cell layers. From the work of others, purinergic mechanisms are known to alter aqueous humor inflow and intraocular pressure. He specifically proposes that: (1) ATP (and possibly other nucleotides and adenosine) are released directly by NPE and PE cells; (2) Membrane ecto-enzymes hydrolyse ATP to AMP and extracellular adenosine; (3) Adenosine acts on NPE cells to stimulate aqueous humor formation by activating Cl- channels; (4) Extracellular ATP stimulates aqueous humor reabsorption by activating PE anion channels; and (5) Net aqueous humor formation is regulated by coordinating these opposing purinergic effects (and other less well defined transport effects) on unidirectional secretion and reabsorption. This coordination may reflect synergistic effects of purines and an unidentified intracellular regulator, whose action is mimicked by tamoxifen. The PI will address the following specific aims: (i) What are the characteristic biophysical and pharmacologic properties of the channels targeted by purines, especially the NPE Cl- channels and PE anion channels? (ii) What are the mode and trigger for purine release by the NPE and PE ciliary epithelial cells? (iii) What are the receptors and signaling pathways underlying purinergic stimulation of transport by the NPE and PE cell? (iv) How are these purinergic effects coordinated to modulate net aqueous humor secretion? The proposed interdisciplinary research will combine patch-clamp and volumetric physiologic methods with molecular biological and structural strategies, correlating measurements from isolated-cell, broken-cell and cell-couplet preparations with those from whole tissues. The novel hypothesis (that aqueous humor formation is directly modulated by an interlayer paracrine release of purinergic messengers) can provide the basis for understanding the purinergic effects on aqueous humor dynamics.